Nitric oxide (NO) is an important signaling molecule that regulates diverse functions relevant to cardiovascular function, apoptosis and angiogenesis. NO is best known for its ability to stimulate soluble guanylyl cyclase (sGC) to produce cGMP and stimulate its downstream signaling pathways. However, NO can also covalently modify cysteines via S-nitrosation (addition of a NO moiety to the cysteine of a protein, SNO). Although this reversible post-translational modification is increasingly recognized as an important regulatory mechanism of protein function, and to play a role in cardiac protection, dynamic regulation of protein nitrosation specificity is poorly understood. Our collaborative team has made the exciting observation that sGC, the key NO receptor, modulates the level of nitrosation of specific proteins in cardiomyocytes and smooth muscle cells. Preliminary data showed that sGC increases nitrosation by a protein-protein interaction-driven SNO transfer (transnitrosation). Moreover, this increased nitrosation is due, for a specific subset of proteins, to the association of sGC with thioredoxin 1 (Trx1), a cardiac protective thiol-redox protein with both transnitrosation and denitrosation activities. Initial mass spectrometry and biochemical analyses showed that sGC transnitrosates Trx1, which in turn nitrosates a specific subset of targets, a finding supported by shTrx1 knockdown experiments in cardiomyocytes. These novel observations lead to the provocative idea that sGC modulates S-nitrosation specificity via a transnitrosation cascade that includes an S-nitrosated Trx1 intermediate. This study aims to answer three critical questions based on this hypothesis. Aim1: What is the mechanism of sGC transnitrosation of Trx1? We will identify key cysteines (Cys) responsible for sGC transfer of SNO to Trx1 and for interaction via mutagenesis and biochemical analyses. Aim2: What are the specific targets of the sGC/Trx1 transnitrosation cascade and the mechanisms underlying target specificity? Using novel and highly specific proteomics approaches, we will quantify the SNO-proteomes modulated by the sGC/Trx1 transnitrosation cascade under nitrosative and oxidative conditions and determine consensus sequence motifs among the target proteins. Aim3: Is sGC-mediated transnitrosation an anti-apoptotic mechanism? NO signaling and Trx1 are crucial components of the anti-apoptotic response to stress. Among the sGC/Trx1 transnitrosation targets identified is the chloride intracellular channel 4 (CLIC4), a regulator of apoptosis, whose nuclear translocation is modulated via specific nitrosation. We will determine whether sGC/Trx1 transnitrosation of CLIC4 is an important inhibitory mechanism of angiotensin II-induced apoptosis of cardiomyocytes, underlying the potential role of this newly discovered transnitrosation cascade in cardiac remodeling following heart failure. This multi-PI project could lead to the discovery of novel cardioprotective pathway driven by specific S-nitrosation.